Competition between Elastase and Related Proteases from Human Neutrophil for Binding to α1-Protease Inhibitor

Abstract

The protease-antiprotease imbalance that is characteristic of most inflammatory lung disorders depends on the spatial-temporal regulation of active inhibitor and protease concentrations in lung secretions. We have studied the competition between the three main serine proteases from human neutrophil primary granules in their binding to alpha1-Pi, the main serine proteases inhibitor in lung secretions. Elastase was the only target of alpha1-Pi when identical molar amounts of purified inhibitor and the three proteases were tested together. The other two proteases were only inhibited once elastase was saturated. Elastase remained the preferred target of inhibitors when bronchoalveolar lavage fluids from patients with lung pneumonia and acute respiratory distress syndrome were used as the source of inhibitors, in spite of the presence of additional inhibitors in lung secretions. Since neutrophil proteases are expressed at the neutrophil surface, we also measured residual activities of membrane-bound proteases after purified neutrophils were incubated with bronchoalveolar fluids. Again, elastase was the preferred target of the inhibitors. We conclude that protease 3 and cathepsin G are not controlled as efficiently as elastase in lung secretions, a feature that must be taken into account when developing inhibitor-based anti-inflammatory therapies.

Inhibition of Neutrophil Elastase by α1-Protease Inhibitor at the Surface of Human Polymorphonuclear Neutrophils

“…Our finding that stoichiometric amounts of ␣1-Pi almost completely inhibit mHNE agrees with our recent finding that the inhibitor binds essentially to HNE, and not to Pr3 or cathepsin G, although these potential targets for this inhibitor are also present at the cell surface (26). This is explained, at least in part, by the different rate constants for association and by the two-step mechanism of Pr3 inhibition that could favor HNE binding when competition occurs (47).…”

“…No HNE activity was detected in crude BALF, suggesting that both free HNE and mHNE are controlled by endogenous inhibitors, as previously shown by Western blotting (26). However, the BALF neutrophils were morphologically identical with activated blood neutrophils, and flow cytometry showed thet they bore CD63 on their FIGURE 4.…”

“…The protocol was approved by the local institution Ethics Committee. BAL was performed, as previously reported (26). BALF samples were collected under sterile conditions, and the fifth fraction, which was not contaminated with epithelial or bronchial cells, was filtered and retained for biochemical and enzymatic analysis (26).…”

Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases

“…␣1-PI is an excellent irreversible inhibitor of HNE that also inhibits related neutrophil proteases PR3 and CG. ␣1-PI inhibits HNE with a inhibitor/enzyme stoichiometry of 1, whereas that with PR3 and CG is slightly higher because of partial proteolytic inactivation of some inhibitor molecules occurring during the course of the interaction (Duranton and Bieth, 2003;Korkmaz et al, 2005b). The second-order constants of association of ␣1-PI with HNE, PR3, and CG are 6.5 ϫ 10 7 , 8.1 ϫ 10 6 , and 4.1 ϫ 10 5 M Ϫ1 ⅐ s…”

Proteinase 3 activity in sputum from subjects with alpha-1-antitrypsin deficiency and COPD

“…However, selective inhibitors of NE have not been fully effective in controlling neutrophil-mediated damage in the airways [11], supporting the likelihood that other proteinases play a role. Further, KORKMAZ et al [12] have suggested that A1AT preferentially inhibits NE, and that PR3 is only inhibited after NE is completely neutralised, suggesting that PR3 may play a more important role when both are released.…”